Because the study of many lipid-mediated second messengers requires rapid reproducible and exquisitely sensitive analysis, Electrospray ionization mass spectrometry techniques will be developed to directly analyze the phospholipid class, subclass and molecular species distribution of cell extracts to facilitate fast and precise analysis without prior HPLC purification. Tandem mass spectrometry will be performed to determine the individual molecular species and fractional distribution of isobaric ion peaks of membrane extracts. Through utilization of methods developed herein, the molecular species selectivity of the 30 kDa and the 85 kDa intracellular phospholipase A2 in their naturally occurring membrane environments and platelet phospholipid alterations after thrombin stimulation will be examined by electrospray mass spectrometry utilizing purchased phospholipases A2.